I.INTRODUCTION
Human DEK gene on chromosome 6p encodes a 43kD nuclear phospoprotein that was originally identified as part of a fusion protein found in a subset of acute myeloid leukemia carrying a t(6;9) translocation1). DEK has been found to function as an architectural protein, a majority of which is bound to chromatin, and to regulate DNA replication, transcription, DNA repair, as well as RNA splicing2). According to several in vitro studies, DEK overexpression may play a role in tumor development through suppressing cellular senescence, apoptosis, and differentiation, which result in promoting cell growth and survival3,4).
DEK upregulation has been described in a number of human malignancies, regardless of t(6;9) translocation, such as hepatocellular carcinoma, glioblastoma, ovarian carcinoma, breast carcinoma, cervical carcinoma, melanoma, Merkel cell carcinoma, and colorectal carcinoma5-11). In particular, DEK overexpression in bladder cancer and retinoblastoma has been associated with the gain of chromosome 6p22 that contains the DEK gene12,13).
DEK expression has been shown to be correlated with various clinicopathological parameters in several cancers. A significant correlation of DEK expression with high histologic grade has been reported in breast, ovarian and colorectal cancer7,9,11). Also, DEK was significantly expressed in the tumors with lymph node metastasis and/or advanced clinical stage9,11). In the area of oral cancer, however, there has been only one report demonstrating upregulation of DEK in tobacco chewingmediated oral squamous cell carcinoma (OSCC)14). They just focused on the identification of differential expression of DEK at the mRNA level between tumor tissues and normal mucosa. To our knowledge, no previous report has evaluated the expression of DEK protein in OSCC tissue samples and its clinicopathological significance. Therefore, our aims in the present study were to determine DEK expression in tissue samples of normal oral mucosa, epithelial dysplasia, and OSCC by immunohistochemistry, to analyze the correlation between DEK expression and clinicopathologic parameters, and to evaluate the value of DEK as a prognostic marker for patient’s survival.
II.MATERIALS AND METHODS
1.Patients and tissue samples
Ten normal oral mucosa samples, 10 epithelial dysplasia, and 60 OSCC samples were studied by immunohistochemistry. All tumors were surgically removed at the Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital. Of the 60 OSCC patients, 49 were male, and the mean patient age was 56 years (ranged from 21 to 86 years). The patients’clinical information, including age, gender, tumor size, lymph node metastasis, recurrence, and TNM stage, is summarized in Table 1. Tumors were staged according to the TNM classification as recommended by the American Joint Committee on Cancer (AJCC).
2.Immunohistochemistry
Formalin-fixed, paraffin-embedded sections (4μm) were used for immunohistochemical staining. The sections were deparaffinized through a series of xylene baths and then rehydrated in graded alcohols. Endogenous peroxidase activity was blocked by incubating the sections with 3% hydrogen peroxide for 10 minutes at room temperature. For antigen retrieval, the sections were treated with target retrieval solution, pH 9.0 (DAKO, Glostrup, Denmark) in the microwave for 10 minutes. Sections were incubated in 10% normal goat serum for 30 minutes to reduce non-specific staining. Sections were then incubated with a rabbit polyclonal anti-human DEK antibody (1:200, ab22826; Abcam, Cambridge, UK) for 1 hour at room. The slides were stained using the DAKO EnVision kit. Immunohistochemical reactions were developed with diaminobenzidine (DAB) as the chromogenic peroxidase substrate, and slides were counterstained with Mayer’s hematoxylin. Breast cancer tissue was used as a positive control. As a negative control, the primary antibody was replaced with PBS.
3.Evaluation of immunohistochemical staining
DEK expression was evaluated independently by two investigators who scored staining using semiquantitive immunoreactive scores. Only the nuclear expression was considered as positive staining. The immunostaining for DEK was scored as 0 (no or < 5% positive cells), 1 (5-25% positive cells), 2 (26-50% positive cells) or 3 (>50% positive cells)8). Cases with a score ≥ 2 were defined as high expression, whereas cases with a score less than 2 were interpreted as low expression.
4.Western blot analysis
A total of 13 OSCC cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM), a mixture of DMEM and F12 or RPMI 1640, supplemented with 10% FBS and 1% antibiotic antimycotics. All cell lines were cultured at 37°C in a humidified atmosphere of 5% CO2. Whole cell lysates were harvested with RIPA buffer supplemented with protease inhibitor cocktail (Sigma-Aldrich, MO, USA). The BCA protein assay kit (Pierce Biotechnology, Rockford, USA) was used to determine the concentration of protein. Samples (30μg) were mixed with sample buffer, incubated at 95°C for 5min, and electrophoresed on 12% SDS/ polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (Pall, MI, USA) and probed with antibody to DEK (1:1000, BD Biosciences, San Jose, CA).
5.Statistical analysis
All of the statistical analyses were performed using the IBM SPSS statistics 20 (IBM Corporation, NY, USA). Correlation between DEK expression and clinicopathological parameters were analyzed by chi-square test and Fisher’s exact tests. The survival rates were calculated using the Kaplan- Meier method and analyzed using the log-rank test. P<0.05 was considered statistically significant.
III.RESULTS
1.DEK protein expression in normal oral mucosa, epithelial dysplasia, and OSCC tissue samples
Nuclear expression of DEK protein was detected in both normal oral mucosa and OSCC tissues. In the normal oral mucosa, DEK expression was detected mainly in the nuclei of the basal and parabasal layers of epithelium (Fig. 1A). However, DEK was more diffusely expressed up to the superficial layer of epithelium in the epithelial dysplasia (Fig. 1B). Significant increase of DEK expression was found in early invasive tumor area when compared to the adjacent dysplastic epithelium (Fig. 1C). 68.3% of OSCC cases showed high expression (score ≥ 2) of DEK protein (Fig. 1D).
2.Correlation between DEK expression and the clinicopathological parameters of OSCC
Table 2 shows the correlations between DEK expression and clinicopathological parameters. Statistical analysis revealed that DEK overexpression in OSCC was positively correlated with high histologic grade, presence of lymph node metastasis, and advanced clinical stage. DEK expression was more highly expressed in moderately and poorly differentiated OSCC than in well-differentiated cases (P=0.001). Also, high expression of DEK protein was more frequently detected in the OSCC samples with lymph node metastasis (P=0.003) and advanced clinical stage (P=0.039). However, there was no correlation between DEK expression and age, gender, tumor size, or recurrence.
In the Kaplan-Meier survival analysis, there was significant difference in overall survival between the patients with high and low expression of DEK (P=0.019, Fig. 2)
3.Expression analysis of DEK in OSCC cell lines
Expression of DEK protein was evaluated in 13 OSCC cell lines by western blot analysis. All cell lines expressed DEK protein to varying degree. Relatively strong expression of DEK was detected in HSC-3 and Ca9-22 cell lines (Fig. 3).
IV.DISCUSSION
Oral cancer is the eleventh most common cancer in the world, with an estimated 300,000 new cases and 145,000 deaths annually15). The majority of oral cancers are squamous cell carcinoma. Despite of advances in understanding of the underlying molecular pathogenesis of OSCC, it remains a lethal disease in over 50% of the cases because of late detection of advanced stage cancer. Therefore, developing possible new therapeutic molecular targets is of importance to improve patient’s prognosis.
Many researches have supported that DEK is a bona fide oncogene that play a role in central pathway to promote tumor growth and survival. Although DEK upregulation has been described in a number of human malignancies, oncogenic DEK activities have been more elucidated by Wise-Draper and his colleagues. They demonstrated that DEK overexpression promotes epithelial transformation and DEK knock mice could not form benign papilloma in a classical chemical carcinogenesis model4). These results support the oncogenic abilities of DEK more directly, in addition to their previous findings demonstrating that DEK suppresses senescence, apoptosis, and epithelial differentiation2- 4). In their report, DEK depletion by RNA interference resulted in apoptosis of cancer cells both in vivo and in vitro4), suggesting that targeted suppression of DEK might be a possible new treatment strategy to suppress cellular transformation and induce apoptosis of tumor cells in human cancer.
In the present study, DEK expression tended to increase through the full thickness of epithelium in the dysplastic oral mucosa when compared with normal epithelium which showed positivity restricted to basal and parabasal layers. In addition, significant increase of DEK expression was found in early invasive tumor area compared to the adjacent dysplastic epithelium. Similar results are also found in cervical, colorectal, and gastric cancer8,11,16). DEK expression in dysplastic lesion and cancer was significantly higher than that in the adjacent normal mucosal of cervix, colon, and stomach. Also, DEK expression was found to be a tumor marker that can distinguish benign nevi from malignant melanoma17). These results suggest that DEK may play a role in the early stage of carcinogenesis in several types of organs. Therefore, it is needed to qualify the value of DEK expression as a biomarker for early diagnosis of OSCC.
DEK expression has been associated with several clinicpathological parameters in diverse malignancies. In consistent with our result, a significant correlation of DEK expression with high histologic grade was found in breast, cervical, ovarian, colorectal, and gastric cancer7-9,11,16). It means that DEK expression could aid in gauging the differentiation potential of tumor cells. In line with the previous results in breast, colon, and stomach9,11,16), DEK was significantly expressed in OSCC with lymph node metastasis and advanced clinical stage. In addition, we found that patient with high DEK expression had worse survival rate than those with low expression as like other reports in colorectal and gastric cancers11,16). Therefore, DEK expression may be also a reliable biomarker to predict the prognosis in OSCC.
Despite the results showing strong association between DEK expression and cancer, molecular mechanisms underlying tumor progression by DEK are still unclear. It was known that DEK is an inhibitor of p53-dependent and -independent cellular senescence and apoptosis2,4), and transcriptionally upregulated by the p16-pRb-E2F pathway18). Recently, Lin et al. studied the mechanism by which DEK overexpression regulate cancer growth and apoptosis in colorectal cancer19). They demonstrated that upregulation of DEK was involved in the p53/MDM, Bcl-2/Bax, and caspase pathway. When the expression of DEK was suppressed, the expression levels of mutant-p53 and MDM2 decreased greatly and Bcl-2/Bax ratio was also significantly reduced so that it could induce the activation of caspase pathway for apoptosis. Taken together, DEK could play a role in tumor progression by inhibiting cellular senescence, enhancing the proliferation, and inhibiting the apoptosis of cancer cells using those signaling pathways. Further investigation is needed to identify another molecular pathway to control the oncogenic role of DEK.
In conclusion, we investigated the expression and clinopathological significance of DEK in OSCC. Significant DEK expression was found in OSCC tissue samples and then high expression of DEK significantly correlated with the histologic differentiation, LN metastasis, and clinical stage. Patients with high expression of DEK showed worse overall survival rate than those with low expression. Our results suggest that DEK may play a role in tumor progression of OSCC and could be a possible prognostic factor for patient with OSCC.
